Erbin as a negative regulator of Ras-Raf-Erk signaling

ABSTRACT

The instant invention provides a method of inhibiting proliferation of maligant cells by administration of a Ras-activity inhibitory effective amount of DNA which encodes the protein Erbin into the maligant cells.  
     The invention also provides methods for evaluating proliferative properties and progression of proliferative activities in tumor cells by measurement of Erbin in tumor tissue.

[0001] This application takes priority from Provisional PatentApplication No. 60/383,603 filed May 29, 2003.

BACKGROUND AND FIELD OF THE INVENTION

[0002] This invention relates to use of Erbin as a novel suppressor ofRas for inhibition of cell, particularly malignant cell, proliferation.Erbin has now been found to be a negative regulator of the Ras-Raf-Erksignaling pathway.

[0003] Extracellular signal regulated kinases (Erk) are a subfamily ofmitogen-activated protein kinases (MAPK) that play important roles in agreat array of cell programs, including proliferation, differentiationand apoptosis. As exemplified by binding to growth factors such as EGF,receptor typrosine kinases are activated and undergo autophophorylationon tyrosine residues. The phosphorylated tyrosine residues recruitadaptor proteins to plasma membrane by directly interacting with modulesincluding Sre homology 2 (SH2) or phosphotyrosine binding domain (PTB).Grb2, one such adaptor, brings guanyl nucleotide exchange factor (SOS)to the plasma membrane in proximity with Ras and expedites exchange ofGDP and GTP on Ras. The activated Ras (GTP-bound) then directly binds toRaf and allows the latter to be activated at the plasma membrane. ActiveRaf triggers sequential activation of MEK, a MAPK kinase, and Erk,leading to phosphorylation of various regulatory proteins, includingnuclear transcription factors such as Erk-1 and Myc as well as manycytoplasmic proteins.

[0004] In the past, extensive efforts have been made to identify factorsthat participate in the regulation of Ras-Raf-Erk pathway. Severalmodulators have been identified that positively influence the pathway atdifferent levels. For example, MEK partner 1 (MP1) was isolated as abinding protein that interacts with both MEK1 and Erk 1 to enhance theefficiency of Erk phosphorylation by MEK. A second protein is the KinaseSuppresor of Ras (KSR) that is believed to act as a scaffold for Raf-1,MEK and Erk instead of a Raf kinase although it contains a kinasedomain. A third such regulator is the Connector Enhancer of KSR (CNK)with multiple functional domains that directly binds to Raf and isinvolved in activation of the Raf/MEK/Erk pathway. Another interestingprotein is Sur-8 that contains multiple leucine-rich regions (LRR) andbinds to both Ras and Raf-1. Although Ras can directly associate withRaf when it is activated by charging with GTP, the presence of Sur8increases the interaction between Ras and Raf and the activation ofdownstream signaling events. These non-enzymatic factors are importantregulators for normal cell proliferation and differentiation.

[0005] In addition, there are negative regulators of the Ras pathway incells. Sprouty, a Ras suppressor in Drosophila and its mammalianhomologue Spred (Sprouty-related EVH1 domain-containing protein) appearto serve as physiological negative feedback regulators of growthfactor-mediated Erk pathway. The Ras effector RINI has been shown toinhibit Ras-induced activation of Raf by competitively binding to activeRas. Additionally, the Raf kinase inhibitory protein (RKIP), initiallyisolated as a phosphatidylethanolamine binding protein, binds directlyto the kinase domains of both Raf and MEK and inhibits MEKphosphorylation. These negative regulators function to ensure that allprograms are adequately executed through autonomous turn-on and turn-offmechanisms. They may also counterbalance over-amplified proliferativesignals caused by active mutation of Ras frequently occurring in humancancers or mutation of upstream components leading to the increasedactivity of the wild-type Ras which is found as well in human cancers.Such an inhibitory mechanism is key to maintain normal cell growth rate.

[0006] Erbin belongs to the LAP (LRR and PDZ) protein family of PDZdomain-containing proteins. In addition to Erbin, the family membersinclude LET-413 in C. elegans, Scribble, a Drosophila protein essentialfor epithelial integrity, Densin-180, and Lano in mammals. Geneticstudies in non-vertebrate have demonstrated that LAP proteins play arole in cell polarity and cell morphology of epithelial cells. Erbin wasinitially identified as a binding partner for ErbB2 and delta-cateninand ARVCF. This 180-kDa protein contains two domains: LRR and PDZ. Thesequence of Erbin has been deposited in GenBank and assigned No. AF263744. The biological function of Erbin and its impact onErbB2-mediated signaling have yet to be addressed.

SUMMARY OF THE INVENTION

[0007] The instant invention provides a method of inhibitingproliferation of maligant cells by administration of a Ras-activityinhibitory effective amount of DNA which encodes the protein Erbin intothe maligant cells. The amount of DNA administered is sufficient toprovide a blood concentration of 1 μM to 100 μM of DNA which encodesErbin into the cells. The DNA encoding Erbin may be administered viaviral vectors.

[0008] The invention also provides methods for evaluating proliferativeproperties and progression of proliferative activities in tumor cells bymeasurement of Erbin in tumor tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0009] This invention relates to use of Erbin, a known protein whosesequence is known and a DNA sequence encoding Erbin is deposited inGenBank as No. AF 263744, which is: aaagatcttt tttttttttt tttctttttttttcggcgga gatcctcgtt (SEQ ID. No. 1) ggggctggga aactcctgca aaactcgagaccaggaagcc agcccgcacc ccaaccccca ccaaagccac ctactcttct tctgtgggaggccagtccac atccgctctc acccgagaga gatattcagc tggatccaaa gtgactgatgaagggaagga aatcatgtca agcgaagcct tgaaaaagct gccctgagac ggtgtcccgccgaaagaatg ttggctcaat taagaaacat cagggagata aattcaaccc agtgtgtctaaaaatgacta caaaacgaag tttgtttgtg cggttggtac catgtcgctg tctacgaggggaagaggaga ctgtcactac tcttgattat tctcattgca gcttagaaca agttccgaaagagattttta cttttgaaaa aaccttggag gaactctatt tagatgctaa tcagattgaagagcttccaa agcaactttt taactgtcag tctttacaca agctgagttt gccagacaatgatttaacaa cgttaccagc atccattgca aaccttatta atctcaggga actggatgtcagcaagaatg gaatacagga gtttccagaa aatataaaaa attgtaaagt tttgacaattgtggaggcca gtgtaaaccc tatttccaag ctccctgatg gattttctca gctgttaaacctaacccagt tgtatctgaa tgatgctttt cttgagttct tgccagcaaa ttttggcagattaactaaac tccaaatatt agagcttaga gaaaaccagt taaaaatgtt gcctaaaactatgaatagac tgacccagct ggaaagattg gatttgggaa gtaacgaatt cacggaagtgcctgaagtac ttgagcaact aagtggattg aaagagtttt ggatggatgc taatagactgacttttattc cagggtttat tggtagtttg aaacagctca catatttgga tgtttctaaaaataatattg aaatggttga agaaggaatt tcaacatgtg aaaaccttca agacctcctattatcaagca attcacttca gcagcttcct gagactattg gttcgttgaa gaatataacaacgcttaaaa tagatgaaaa ccagttaatg tatctgccag actctatagg agggttaatatcagtagaag aactggattg tagtttcaat gaggttgaag ctttgccttc atctattgggcagcttacta acttaagaac ttttgctgct gatcataatt acttacagca gttgcccccagagattggaa gctggaaaaa tataactgtg ctgtttctcc attccaataa acttgagacacttccagagg aaatgggtga tatgcaaaaa ttaaaagtca ttaatttaag tgataatagattaaagaatt taccctttag ctttacaaag ctacagcaat tgacagctat gtggctctcagataatcagt ccaaacccct gatacctctt caaaaagaaa ctgattcaga gacccagaaaatggtgctta ccaactacat gttccctcaa cagccaagga ctgaggatgt tatgtttatatcagataatg aaagttttaa cccttcattg tgggaggaac agaggaaaca gcgggctcaagttgcatttg aatgtgatga agacaaagat gaaagggagg cacctcccag ggagggaaatttaaaaagat atccaacacc atacccagat gagcttaaga atatggtcaa aactgttcaaaccattgtac atagattaaa agatgaagag accaatgaag actcaggaag agatttgaaaccacatgaag atcaacaaga tataaataaa gatgtgggtg tgaagacctc agaaagtactactacagtaa aaagcaaagt tgatgaaaga gaaaaatata tgataggaaa ctctgtacagaagatcagtg aacctgaagc tgagattagt cctgggagtt taccagtgac tgcaaatatgaaagcctctg agaacttgaa gcatattgtt aaccatgatg atgtttttga ggaatctgaagaactttctt ctgatgaaga gatgaaaatg gcggagatgc gaccaccatt aattgaaacctctattaacc agccaaaagt cgtagcactt agtaataaca aaaaagatga tacaaaggaaacagattctt tatcagatga agttacacac aatagcaatc agaataacag caattgttcttctccatctc ggatgtctga ttcagtttct cttaatactg atagtagtca agacacctcactctgctctc cagtgaaaca aactcatatt gatattaatt ccaaaatcag gcaagaagatgaaaatttta acagcctttt acaaaatgga gatattttaa acagttcaac agaggaaaagttcaaagctc atgataaaaa agattttaac ttacctgaat atgatttgaa tgttgaagagcgattagttc taattgagaa aagtgttgac tcaacagcca cagctgatga cactcacaaattagatcata tcaatatgaa tcttaataaa cttataacta atgatacatt tcaaccagagatcatggaaa gatcaaaaac acaggatatt gtgcttggaa caagcttttt aagcattaattctaaagagg aaactgagca cttggaaaat ggaaacaagt atcctaattt ggaatccgtaaataaggtaa atggacattc tgaggaaact tcccagtctc ctaataggac tgaaccacatgacagtgatt gttctgttga cttaggtatt tccaaaagca ctgaagatct ctcccctcagaaaagtggtc cagttggatc tgttgtgaaa tctcatagca taactaatat ggagattggagggctaaaaa tctatgatat tcttagtgat aatggacctc agcagccaag tacaaccgttaaaatcacat ctgctgttga tggaaaaaat atagtcagga gcaagtctgc cacactgttgtatgatcaac cattgcaggt atttactggt tcttcctcat cttctgattt aatatcaggaacaaaggcaa ttttcaagtt tgattcaaat cataatcccg aagagccaaa tataataagaggccccacaa gtggcccaca atctgcacct caaatatatg gtcctccaca gtataatatccaatacagta gcagtgctgc agtcaaagac actttgtggc actccaaaca aaatccccaaatagaccatg ccagttttcc tcctcagctc cttcctagat cagagagcac agaaaatcaaagttatgcta aacattctgc caatatgaat ttctctaatc ataacaatgt tcgagctaatactgcatacc atttacatca gagacttggc ccagcaagac atggggaaat gtgggccatctcaccaaacg accgacttat tcctgcagta actcgaagta caatccagcg acaaagtagtgtgtcctcca cagcctctgt aaatcttggt gatccaggct ctacaaggcg ggctcagattcctgaaggag attatttatc atacagagag ttccactcag cgggaagaac tcctccaatgatgccaggat cacagagacc cctttctgca cgaacataca gcatagatgg tccaaatgcatcaagacctc agagtgctcg accctctatt aatgaaatac cagagagaac tatgtcagttagtgatttca attattcacg gactagtcct tcaaaaagac caaatgcaag ggttggttctgagcattctt tattagatcc tccaggaaaa agtaaagttc ctcgtgactg gagagaacaagtacttcgac atattgaagc caaaaagtta gaaaagatgc ctttgagtaa tggacagatgggccagcctc tcaggcctca ggcaaattat agtcaaatac atcacccccc tcaggcatctgtggcaaggc atccctctag agaacaacta attgattact tgatgctgaa agtggcccaccagcctccat atacacagcc ccattgttct cctagacaag gccatgaact ggcaaaacaagagattcgag tgagggttga aaaggatcca gaacttggat ttagcatatc aggtggtgtcgggggtagag gaaacccatt cagacctgat gatgatggta tatttgtaac aagggtacaacctgaaggac cagcatcaaa attactgcag ccaggtgata aaattattca ggctaatggctacagtttta taaatattga acatggacaa gcagtgtcct tgctaaaaac tttccagaatacagttgaac tcatcattgt acgagaagtt tcctcataag cactgtggac aaaaaaagcggggaagacag caagatttat tggaagatac ttacagggga aattaatatt ttgactatttttatatataa agaagaactc aaaaaattat gttcaaattt gtacattaat gaaataatggataaaggaga ctgttgaatt cataccatat aaaacttgtt aggtttttaa acatagcaatcaaggctaca aaaacaaacc tgtgttgttt ttgtatagat tgtaggttta tttttggatttcatatacat gactgaactg tgtgcaaggc aatagttagc cttgatttta gcccagagacagatggcaga gctatctctc tcatagcttt tatgccctta tttttattca actggtattaatgtttttct cctgaaacta ctttttttga tgtgggcaag agatttgaag tgttggcttttgctatgtgc atattgaatt gaagagtgag taggtgaagg tggtgctggt gggttcactttccaaggcca gactaaaaca gttattttct ataaaaatct ggaagcaaag aatggggatggggagagcta cgtggtagta tgtttttatt aggagaataa tgcaataaaa tatgtaatgtcttttttata aagcaaaaaa gacaataatt gcatttatga gctcggcagg atctgttcttgtcatagcca ttgactatac atttgctact ggtgattcag tttttaattt tttagtcacaggaaattttt aactctactg tagatgcatg tccatgcatt ttctgtgtta tggaaatccactgatttttt tttttttttc aaatggtggt acttgcaatc tgttttataa ttagtgctccatttaaatct aatttataat ttttatttta agcagcaaat gaaacaaaaa tggccagttttaagattgtg ttgcctgtaa cacaaaatgt tacgaaggtt taggaaagcc tctttgatttttgtttggcc ttgcattggc ccttggtaaa gtaaaaggaa acagtacact tggagctaggaaaccaaagc aagctttgtg aaactggcac agtgatagag aattgctgtg gagagttatagagcaaaggg atgggtcctt gaggcctgcc agtgtgtaaa ggtgttcaaa taaagggctgtttctacagg taacattaaa tgtgaactca acacttccag agtctttaaa gggtttctatgtgtatcagt gtaatagtgt tttaccacca actgcctttc tttgttccta gttactgtaacaaatatttg atgatagagg tttattaatt ttgtttatcc agaccattaa ttttatttgtttttgtctat gtaatcaaat aaaatttgag taacatgtaa tggtaaggat taatgcatggttatttggac cagaaaaaag tgccatagaa gaccaataac tgtttagttg aggctagtctggaacctttc attagagcaa tatttggtta ttgcacttca tttttattta ctaagaaatgcaatttggga atttttaatc tgttatgctt tgtttatcaa ccttgatttt aattaagacttttataagac tagcttaaaa caccaaccaa cattattttt gcaaaagtga gttggactcactttccattc ttgctagtca gagtaagtag gcagcacttt taaaaatatg tgaactcaaatattgcactt ctttcaagat gttatcaatt ggttattgta ctgtatagtt ttaataattttgattgaaac cctttaacaa ctctttgtaa attttaactc attttagttg attttcagtactatttacat aggaattgat ttttatggat atagtagaag aaatgtgctg tattttgataaaattcactt attgtatgtg tgttgtaatc taaaaaaaaa aagaatgaca aacagcttctttaagacaaa aaaaaaaaaa aaaaaaaaa

[0010] Erbin is a negative regulator of the Ras-Raf-Erk signalingpathway. Expression of Erbin decreases neuregulin-induced transcriptionof AChR ε-subunit gene, an event that requires Erk activation. Althoughit interacts with the ErbB2 C-terminus through the PDZ domain, Erbin hasno effect on ErbB2 tyrosine phosphorylation and binding to adaptorproteins Shc or Grb2. In contrast, expression of Erbin greatly impairsErk activation by ligands that activate receptor tyrosine kinases,without causing a significant change in AKT activity. It is now shownherein that Erbin diminishes the ability of Ras but not Raf-1 toactivate Erk. Consistently, Erbin binds only to active Ras, as opposedto inactive Ras or Raf, resulting in inhibition of the interactionbetween Ras and Raf both in vivo and in vitro. As a suppressor of theRas by disrupting Raf binding to active Ras, Erbin acts to suppresstumorigenesis of cancer cells, especially breast and prostate cancercells. The control of tumorigenesis by increasing level of Erbin inmalignant tissue by various means, especially through gene therapy, toobtain a blood or target tissue cell concentration of about 1 μM to 100μM concentration would be appropriate for control of malignancies.

[0011] Materials and Methods:

[0012] Plasmid Construction—The human Erbin N-terminal domain (aa 1-391)consisting of 16 LRRs was generated by PCR amplification using senseprimer containing BamHI and antisense primer containing XhoI. Theresulting 1.2 kb-fragment was digested with BamHI and XhoI, andsubcloned in the BamHI-SalI sites of yeast vector pGBT9 downstream ofthe Gal4 DNA binding domain (Clontech). Myc-Erbin LRR, Myc-ErbinD965 andMyc-DPDZ were generated by introducing a stop codon after the LRR domainfollowing aa 965 or aa 1279 in pRK5-Myc-Erbin. The N-terminal deletionmutant (pRK5-Erbin965) was described previously (Huang, Y. Z., Wang, Q.,Xiong, W. C., and Mei, L. (2001) J Biol Chem 276, 19318-19326); andBorg, J. P., Marchetto, S., Le Bivic, A., Ollendorff, V.,Jaulin-Bastard, F., Saito, H., Fournier, E., Adelaide, J., Margolis, B.,and Birnbaum, D. (2000) Nat Cell Biol 2, 407-414). A fragment encodingthe full-length Akt cDNA generated by PCR using sense primer containingEcoRI and antisense primer containing XhoI. The resulting 1.6kb-fragment was digested with EcoRI and XhoI, and subcloned intoEcoRI-XhoI sites of the mammalian expression vector pCS2+MT (for the Myctag at the amino terminus). Wild type-ErbB2 and constitutive active formof ErbB2 (NeuT) (generously provided by Dr. M. C. Hung, University ofTexas M. D. Anderson Cancer Center) were subcloned downstream of theFlag-tag and an artificial signal peptide in pCMV. pCMV-Flag-Erk1 wasgenerously provided by Dr. Mike Weber (University of Virginia).Flag-Ras, Flag-RasV12, Flag-RasN17, GST-Raf-C4, and GST-Raf-BXB weredescribed as previously (Zang, M., Hayne, C., and Luo, Z. (2002) J BiolChem 277, 4395-4405).

[0013] Cell Culture and Transfection—HEK 293 cells and COS-7 cells werecultured as described previously (Huang, supra). The C2C2 cells weremaintained as undifferentiated myoblasts in DMEM with high glucosesupplemented with 20% fetal bovine serum, and 0.5% chicken embryoextract. Fusing of myoblasts into myotubes was induced by culturingmyoblasts for 48 hours in differentiation medium DM (DMEM plus 4% horseserum). Mouse lung epithelial Mv1Lu cells were maintained in DMEM plus10% fetal bovine serum (FBS). Rat pheochromocytoma-derived PC12 cellswere grown in DMEM supplemented with 10% FBS and 5% horse serum. HEK293, COS-1 and C2C12 cells were transfected with the standard calciumphosphate technique. PC12 cells and Mv1Lu cells were transfected withSuperFect regents (Qiagen). Two days after transfection, cells werewashed with PBS and lysed in the modified RIPA buffer containing 20 mMsodium phosphate, pH 7.4, 50 mM sodium fluoride, 40 mM sodiumpyrophosphate, 1% Triton X-100, 2 mM sodium vanadate, 10 mMp-nitrophenyl phosphate, and protease inhibitors. Lysed cells wereincubated on ice for 20 min and centrifuged at 13,000×g for 10 min at 4C. The clear supernatant was designated as cell lysates.

[0014] Immunoprecipitation and Immunoblottina—Cell lysates (˜400 μg ofprotein) were incubated without or with indicated antibodies one hour at40° C. and subsequently with protein A- or protein G-agarose beadsovernight at 4° C. on a rotating platform. After centrifugation, beadswere washed five times with the modified RIPA buffer. Bound proteinswere eluted with the SDS sample buffer, resolved by SDS-PAGE andtransferred onto nitrocellulose membranes (Schleicher and Schuell).Nitrocellulose membranes were incubated at room temperature for one hourin the blocking buffer containing Tris-buffered saline with 0.1% Tween(TBS-T) containing 5% milk or 5% BSA followed by an incubation withindicated antibodies in the blocking buffer. After washing 3 times for 5min each with TBS-T, the membrane was incubated with horseradishperoxidase-conjugated donkey anti-mouse or anti-rabbit IgG (AmershamPharmacia Biotech) followed by washing. Immunoreactive bands werevisualized with enhanced chemiluminescence substrate (Pierce). In someexperiments, the nitrocellulose filter was incubated in a buffercontaining 62.5 mM Tris/HCl, pH 6.7, 100 mM β-mer-captoenthanol, and 2%SDS at 50° C. for 30 min, and washed with 0.1% Tween 20 in 50 mM TBS atroom temperature for 1 hr, and reblotted with different antibodies. Thefollowing antibodies were used: Flag (M2, Sigma), Myc (9E10, SantaCruz), phospho-MAPK (Promega), phospho-Akt (Ser473, New England Biolab)and Erbin.

[0015] Luciferase Assay—Myoblasts were co-transfected with or withoutMyc-Erbin, plus the ε-subunit promoter-luciferase transgene thatcontains 416 nucleotides of the 5′UTR of the ε-subunit gene (25) and acontrol plasmid pRL-SV40 (Promega). Twenty four hours aftertransfection, the myoblasts were incubated in DM to induce myotubeformation. Myotube formation was complete 48 hours after switch to DM.The C2C12 myotubes were stimulated with neuregulin at a finalconcentration of 10 Nm at 37° C. for 24 hours. Mv1Lu cells weretransiently transfected with the promoter reporter construct p3TP-Lux,which promoter contains three AP-1 sites and the plasminogen activatorinhibitor-1 (PAI-1) promoter and firefly luciferase. pRL-SV40 thatexpress Renilla luciferase under the control of SV40 promoter wascotransfected as a control to monitor the transfection efficiency.Fourty eight hours after transfection, cells were lysed and activitiesof the two different luciferases were assayed with respective substrateswith a dual luciferase assay kit (Promega).

[0016] Differentiation of PC12 cells—PC12 cells were cotransfected pEGFPwith empty vector pRK5-Myc, Myc-Erbin or its mutants, or Erbin RNAiduplex. Forty eight hours after transfection, PC12 cells were stimulatedby 100 ng/ml or 20 ηg/ml NGF for 2 days. Cells were examined byfluorescence microscopy. Cells with processes 1.5 times longer than thediameter of the cell body were considered to be differentiated.

[0017] Inhibition of Erbin expression by RNAi—The target region of siRNAwas 100 nt downstream of the start codon, which contained approximately50% G/C content. The nucleotide sequence was 5′ UAG ACU GAC CCA GCU GGAA dTdT 3′ (nt 866-884) (Borg, J. P., Marchetto, S., Le Bivic, A.,Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Adelaide,J., Margolis, B., and Birnbaum, D. (2000) Nat Cell Biol 2, 407-414). TheNCBI sequence bank was searched against this segment Cof DNA using theblast program, which revealed no match, suggesting of the specificity oftarget recognition by siRNA. The 21-nucleotide RNAs were chemicallysynthesized by Dharmacon Research Inc. Synthetic oligonucleotides weredeprotected and gel-purified. To demonstrate the silencing effect ofendogenous Erbin expression by siRNA, cells in 60 mm-culture dish wereco-transfected with empty vector pEGFP and with siRNA duplex using theSuperFect kit (Qiagen). Briefly, 2 mg of pEGFP and 30 ml of 20 mM RNAiduplex were mixed with 300 ml of Opti-MEM (GIBCO-BRL). After incubating10 min at room temperature, Opti-MEM was added to obtain a final volumeof 1 ml. Cells were incubated with the mixture for 2-3 hours at 37° C.and 5% CO before the addition of 5 ml of growth medium. Seventy twohours after transfection, cells were resuspended in PBS buffer.GFP-positive cells were collected by fluorescence-activated cell sorting(FACS) analysis. Cells were lysed in modified RIPA buffer and lysateswere subjected to immunoblotting for expression of Erbin. In parallelexperiments, GFP-positive PC12 cells were scored for differentiation.

[0018] Protein Assay—Protein was assayed with Coomassie Protein AssayRegent (Pierce) using bovine serum albumin as a standard.

[0019] Erbin inhibits neuregulin-induced transcription of the AChRε-subunit gene and Erk activation—It was found that transcription ofAChR subunit genes is increased by neuregulin, a ligand that activatesthe ErbB2 receptor tyrosine kinase. Previous studies in this laboratoryand by others have demonstrated that neuregulin-induced AChR expressionrequires ErbB2 tyrosine phosphorylation and activation of theRas-Raf-Erk signaling pathway. It now is seen that Erbin, interactingwith ErbB2, plays a role in regulating neuregulin signaling. To testthis hypothesis, it was useful to examine the effect of Erbin on thepromoter activity of ε416-Luc, a transgene reporter that contains 416nucleotides of the 5′UTR of the -subunit gene. Expression of thistransgene is up-regulated by neuregulin or Ras- or Raf-activation andrequires Erk activation. Unexpectedly, it was found that co-expressionof Erbin inhibited neuregulin-activated expression of the ε416-Luctransgene, suggesting that Erbin may regulate the Erk activation.

[0020] To test this hypothesis, it was useful to characterize effects ofErbin on Erk1 activation in COS-7 cells. Flag-Erk1 was activated inresponse to the growth factor NRG in cells cotransfected with ErbB4.Coexpression of Erbin caused a significant decrease in the level ofphopho-Erk, indicating that Erbin negatively regulate the Ras-Raf-MEKErkpathway. The inhibitory effect of Erbin featured the following: (1) itwas dose-dependent; (2) Erbin did not seem to delay the peak Erkactivation that usually occurred within 5 min of stimulation; (3) theinhibition was not growth factor-specific. In addition, expression ofErbin inhibited EGF- and NGF-induced Erk activation and (4) it was Erkactivation-specific, since expression of Erbin had no apparent effect onNRG activation of Akt. Thus, the results demonstrate that Erbinspecifically inhibits Erk activation with little effect on the PI3kinase pathway.

[0021] To identify the domain that inhibits the Erk activation, weexamined the effect of a series of Erbin mutations. The results revealedthat the inhibition of Erk did not require PDZ domain, which interactswith ErbB2 or the region between the LRR domain and the PDZ domain. Incontrast, deletion of the LRR domain disabled Erbin to inhibit Erkactivation. Furthermore, it was demonstrated that the LRR domain wassufficient to mediate the inhibitory effect. These results are inagreement with the differentiation assay and thus indicates that Erbinplays an important role in regulating the Ras-Raf-MEK-Erk pathway.

[0022] Erbin inhibits Erk activation by Ras—An attempt was made todissect the position for Erbin action by walking upstream of Erk. SinceErbB2 directly interacts with the cytoplasmic domain of tyrosine kinasereceptors, it is possible that Erbin interferes with the tyrosine kinaseactivation and/or subsequent binding to adaptor proteins. To test thesehypotheses, it was decided to take the advantage of NeuT, an active formof ErbB2 (Bargmann, C. I., Hung, M. C., and Weinberg, R. A. (1986) Cell45, 649-657). When expressed in COS1 cells, NeuT wastyrosine-phosphorylated, resulting in an increase in the promoteractivity of p3TP-Lux. When NeuT was immunoprecipitated, association ofShc and Grb2 could be easily detected. Coexpression of Erbin had noeffect on either tyrosine phosphorylation of NeuT or its associationwith Shc and Grb2. However, the NeuT-induced promoter activity of 3TPwas greatly inhibited by Erbin, indicating that the site of Erbin actionis downstream of the adapter proteins. Further examination was conductedto determine whether Erbin inhibits Erk activation by Raf or Ras. Sinceexpression of the active mutant of Raf bypasses the requirement ofupstream components for the MEK/Erk activation, Erbin would attenuatethe Erk activation by active Raf, if it acted downstream of Raf. Theresults showed that coexpression of Erbin with active Raf wasessentially without an effect on the Erk activation. Conversely,inhibition of Erk activation by active Ras was evidently observed. Theseresults indicate that Erbin acts above Raf, likely at the level of Ras.

[0023] Erbin disrupts the Ras-Raf interaction—In considering themechanism of the Erbin-induced inhibition of the Erk pathway, it waspostulated that Erbin inhibits the interaction between active Ras andRaf by competitive binding to either of them, or diminishing theGTP-bound form of Ras. To test this, an active mutant of Ras(Flag-RasV12) was coexpressed with GST-Raf1 into HEK293 cell.Flag-RasV12 was found to copurify with GST-Raf1. In a reciprocal study,Raf1 was detected in the immunoprecipitates of active Ras. Remarkably,coexpression of Erbin decreased the interaction between active Ras andRaf. In an alternative study, the Ras pull-down assay developed by Rooijand Bos in which they exploited the nature of high affinity of GTP-Rasfor the Ras-binding domain (RBD) of Raf-1, as compared with GDP-Ras wasemployed. The RBD (aa 50-150) was expressed as a GST-fusion protein andused to pull down GTP-Ras that had been activated inside cellscotransfected with or without Erbin. In cells transfected with Flag-Ras,GTP-Ras was recovered by the GST-Raf-RBD beads. Expression of Erbininhibited the interaction of Raf with growth factor-activated Ras,accompanied by a decrease in phospho-Erk1. These results indicate thatErbin inhibits the Erk pathway by disrupting the interaction betweenactive Ras and Raf.

[0024] Studies were conducted to determine whether Erbin directly boundto active Ras or Raf in a manner to prevent them from forming a complex.When recombinant Erbin was isolated from HEK293 cells contransfectedwith Raf-1, no association was found between these two proteins. Aparallel experiment was performed with Erbin and Ras. Only the activeRasV12 was co-immunoprecipitated with Erbin, as opposed to the dominantnegative mutant Ras. These clearly indicate that Erbin competes withRaf1 by binding to active Ras.

[0025] Inhibition of NGF-induced PC12 cell differentiation by Erbin—Tofurther study the physiological importance of the Ras inhibition byErbin, the effect of its overexpression on neuronal differentiation ofrat pheochromocytoma (PC12) cells was examined. By chronic incubationwith NGF, these cells differentiated developed sympathetic neuron-likephenotypes. The NGF-treated cells stopped to divide and in turnsdeveloped long, sometimes branched processes. The signaling events havebeen well characterized during NRG-induced differentiation of PC12 cellsin which the Ras-Raf-Erk pathway plays an essential role. The inhibitionof Erk activation by Erbin prompted an extended study to more broadlyexamine its effect on NRG-induced differentiation of PC12 cells.Expression of enhanced green fluorescent protein (EGFP) and the pRK5-Mycempty vector (control) had no apparent effect on the differentiation,whereas Erbin-transfected PC12 cells exhibited altered morphology. Theneurites became shorter and were less branched. Under the controlcondition, cells bearing neurites 1.5 times longer than the cell bodyaccounted for 60±5% of the total cell population, whereas the number ofdifferentiated cells are significantly reduced after Erbin transfection.Ectopic expression of the LRR domain showed similar effect ondifferentiation, suggesting the inhibitory activity was contained inthis domain. In contrast, cells transfected with Δ965 encodingC-terminal domain of Erbin appeared to have normal differentiation.

[0026] To verify the inhibitory effect of Erbin on PC12 celldifferentiation, the effect of suppressed Erbin expression in PC12 cellswas examined. RNA interference (RNAi) techniques were used for thispurpose. As an emerging new technique to diminish expression of specificgenes at a cellular level, sequence-specific double-stranded RNA arefirst employed in evolutionarily diverse organisms including plants,fungi, and metazoans (Hammond, S. M., Caudy, A. A., and Hannon, G. J.(2001) Nat Rev Genet 2, 110-119). Recently, RNAi has been shown tospecifically suppress the expression of endogenous and heterologousgenes in mammalian cell lines. Thus, 21 nucleotide RNAi duplexesdirected against Erbin (nucleotides 866-884) were synthesized andtransfected in PC12 cells to suppress the expression of endogenousErbin. Two days after transfection, cells expressing co-transfected EGFPwere sorted out and analyzed for Erbin expression by Western blot.Expression of Erbin in RNAi-transfected cells was significantlydecreased in comparison with missense RNAi-transfected cells (control).The suppressing effect by Erbin RNAi appeared to be specific since ithad no effect on expression of endogenous Erk1. Moreover, expression ofcotransfected EGFP was unaffected. Having demonstrated that sense RNAiinhibits expression of endogenous Erbin, RNAi-transfected PC12 cellswere challenged with 20 ng/ml NGF to induce differentiation. To capturethe maximal effect of Erbin RNAi, a sub-maximal effective concentrationof NGF was used, as at this concentration, NGF caused differentiation ofonly 25±2% PC12 cells. Remarkably, suppressing Erbin expression by senseRNAi enhanced NGF-mediated cell differentiation (52±2%), while missenseRNAi had no significant effect (18±2%). These data reinforce theconclusion that Erbin inhibits NGF-induced differentiation of PC12cells.

[0027] Preparation and administration of viral vectors. The costructionof adenovirus is carried out according to established protocols (Proc.Natl. Acad. Sci. USA (1998) 95: 2509-2514) using a set of commerciallyavailable plasmids. Briefly, DNA encolding the full length of Erbin orthe LRR domain (Erbin-LRR) is coded into pAdTrack-CMV. Ad-Erbin orErbin-LRR and control AdGFP adenoviruses are generated in BJ5183(available from American Type Culture Collection (ATCC) as depositJHU-18) bacterial cells by homologous recimbination of the viralpAdEasy-1 and pAdTrack CMV-Erbin or Erbin-LRR and pAdTrack-CMV,respectively. Cells available from ATCC under deposit number CRL-1573known as 293 cells are used as hosts for viral productions by knownmethods. (See Neuron. (2002), 35:489-505)

[0028] The adenoviruses expressing Erbin or Erbin-LRR are injected intothe tumor tissue to infect cancer cells, particularly breast or prostatecancer cells.

[0029] Administration of Erbin DNA into tumors. Mice are anesthetizedwith phenobarbital sodium. Tumor tissues are injected with 100 μg ofclosed circular DNA (pcDNA3-Erbin or Erbin-LRR) at concentration of 1.0μg/μl in saline using a syringe with a 27-gauge needle. To enhance thetransfection efficiency, injected tumor tissues are subjected toelectroporation with a pair of tungsten needles inserted into the tumorencompassing DNA injection sites. Electric pulses are delivered using anelectric pulse generator.

[0030] Monitoring Erbin expression. Erbin expression in tumor tissuesand cells may be monitored by two methods: Western blot andimmunohitochemical analysis, both using anti-Erbin antibody.

[0031] Western blot: Tumor tissue or cell lysates (about 400 μg ofprotein) from the suspect tissue and from control, normal tissue areresolved by SDS-PAGE, and transferred into nitrocellulose membranes.Nitrocellulose membranes are incubated at room temperature for one hourin the blocking buffer containing Tris-buffered saline with 0.1% Tween(TBS-T) containing 5% milk followed by incubation with anti-Erbinantibody in the blocking buffer. After washing three times for 5 minuteseach with TBS-T, the membrane is incubated with horseradishperoxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences)followed by washing. Immunoreactive bands are visualized with enhancedchemiluminescence substrate (Pierce).

[0032] Immunohistochemical analysis. Tumor tissues are rapidly dissectedand frozen in isopentane cooled with dry ice. Ten μM sections areprepared using a cryostat, thaw mounted on gelatin-coated slides andstored at −80°C. Sections of tumor tissue sections are incubated with 2%normal goat serum (Vector Laboratories, Burlingame, Calif.) in PBS for 1hour at room temperature to reduce background staining and thenincubated with the affinity-purified and anti-Erbin antibody in 2%normal goat serum in PBS overnight at 4° C. After wiashing the sectionsfive times with PBS, each for 30 minutes, the sections are incubatedwith a flourescein istothiocyanate-conjugated anti-rabbit antibody(Zymed Laboratories Inc., San Francisco, Calif.) or visualized by theestablished DAB method. Fluorescent images are captured on Sony CCDcamera mounted on a Nikon E600 microsope using Photoshop imagingsoftware. The amount of Erbin is then determined.

[0033] In each instance, the amount of Erbin as measured is measuredagainst standards and against the amount of Erbin in normal tissues. Itis also possible to evaluate progression of disease by measuring theamount of Erbin in samples from target tissue repeatedly.

[0034] Identification of Erbin mutants. Tumor tissue samples arecollected from patients with breast or prostate cancer and from healthcontrols, matched for geographical origin. DNA is isolated and collectedin tripotassium EDTA strile tubes and immediately stored at −70° C.Genomic DNA extraction is performed on the tissue. The DNA sequenceencoding the LRR domain is subjected to analysis of the Erbin gene. Thepolymorphism is identified using ARMS-PCR as previously described (GenesImmunity (2002) 3:30-33).

[0035] It appears that following Raf activation, Erbin functions to kickRaf off the plasma membrane by competing with Raf in binding to activeRas. Thus, overexpression of Erbin diminishes the accessibility ofRas-GTP to Raf-1. Alternatively, Erbin may compete with Sur-8 forbinding to Ras and then dissociate the Sur 8/Ras/Raf ternary complex,resulting in down-regulation of the Erk pathway. It is conceivable thatErbin may act more quickly and efficiently than Sur 8, as it is enrichedat the plasma membrane. Active mutations of the Ras gene renders it themost frequent oncogene found in human cancers and even more, many otheroncogenes exploit Ras and its downstream cohorts to execute theirfunctions. Thus, the use of Erbin for inhibitory mechanisms and blockersfor this pathway is indicted as a means of inhibiting growth ofmalignant cells. The method involved administration of a proliferationinhibiting effective amount of Erbin or the administration of a vectorwhich gives rise to production of a proliferation inhibiting effectiveamount of Erbin.

1 1 6409 base pairs nucleic acid unknown unknown DNA (genomic) NO NO 1AAAGATCTTT TTTTTTTTTT TTTCTTTTTT TTTCGGCGGA GATCCTCGTT GGGGCTGGGA 60AACTCCTGCA AAACTCGAGA CCAGGAAGCC AGCCCGCACC CCAACCCCCA CCAAAGCCAC 120CTACTCTTCT TCTGTGGGAG GCCAGTCCAC ATCCGCTCTC ACCCGAGAGA GATATTCAGC 180TGGATCCAAA GTGACTGATG AAGGGAAGGA AATCATGTCA AGCGAAGCCT TGAAAAAGCT 240GCCCTGAGAC GGTGTCCCGC CGAAAGAATG TTGGCTCAAT TAAGAAACAT CAGGGAGATA 300AATTCAACCC AGTGTGTCTA AAAATGACTA CAAAACGAAG TTTGTTTGTG CGGTTGGTAC 360CATGTCGCTG TCTACGAGGG GAAGAGGAGA CTGTCACTAC TCTTGATTAT TCTCATTGCA 420GCTTAGAACA AGTTCCGAAA GAGATTTTTA CTTTTGAAAA AACCTTGGAG GAACTCTATT 480TAGATGCTAA TCAGATTGAA GAGCTTCCAA AGCAACTTTT TAACTGTCAG TCTTTACACA 540AGCTGAGTTT GCCAGACAAT GATTTAACAA CGTTACCAGC ATCCATTGCA AACCTTATTA 600ATCTCAGGGA ACTGGATGTC AGCAAGAATG GAATACAGGA GTTTCCAGAA AATATAAAAA 660ATTGTAAAGT TTTGACAATT GTGGAGGCCA GTGTAAACCC TATTTCCAAG CTCCCTGATG 720GATTTTCTCA GCTGTTAAAC CTAACCCAGT TGTATCTGAA TGATGCTTTT CTTGAGTTCT 780TGCCAGCAAA TTTTGGCAGA TTAACTAAAC TCCAAATATT AGAGCTTAGA GAAAACCAGT 840TAAAAATGTT GCCTAAAACT ATGAATAGAC TGACCCAGCT GGAAAGATTG GATTTGGGAA 900GTAACGAATT CACGGAAGTG CCTGAAGTAC TTGAGCAACT AAGTGGATTG AAAGAGTTTT 960GGATGGATGC TAATAGACTG ACTTTTATTC CAGGGTTTAT TGGTAGTTTG AAACAGCTCA 1020CATATTTGGA TGTTTCTAAA AATAATATTG AAATGGTTGA AGAAGGAATT TCAACATGTG 1080AAAACCTTCA AGACCTCCTA TTATCAAGCA ATTCACTTCA GCAGCTTCCT GAGACTATTG 1140GTTCGTTGAA GAATATAACA ACGCTTAAAA TAGATGAAAA CCAGTTAATG TATCTGCCAG 1200ACTCTATAGG AGGGTTAATA TCAGTAGAAG AACTGGATTG TAGTTTCAAT GAGGTTGAAG 1260CTTTGCCTTC ATCTATTGGG CAGCTTACTA ACTTAAGAAC TTTTGCTGCT GATCATAATT 1320ACTTACAGCA GTTGCCCCCA GAGATTGGAA GCTGGAAAAA TATAACTGTG CTGTTTCTCC 1380ATTCCAATAA ACTTGAGACA CTTCCAGAGG AAATGGGTGA TATGCAAAAA TTAAAAGTCA 1440TTAATTTAAG TGATAATAGA TTAAAGAATT TACCCTTTAG CTTTACAAAG CTACAGCAAT 1500TGACAGCTAT GTGGCTCTCA GATAATCAGT CCAAACCCCT GATACCTCTT CAAAAAGAAA 1560CTGATTCAGA GACCCAGAAA ATGGTGCTTA CCAACTACAT GTTCCCTCAA CAGCCAAGGA 1620CTGAGGATGT TATGTTTATA TCAGATAATG AAAGTTTTAA CCCTTCATTG TGGGAGGAAC 1680AGAGGAAACA GCGGGCTCAA GTTGCATTTG AATGTGATGA AGACAAAGAT GAAAGGGAGG 1740CACCTCCCAG GGAGGGAAAT TTAAAAAGAT ATCCAACACC ATACCCAGAT GAGCTTAAGA 1800ATATGGTCAA AACTGTTCAA ACCATTGTAC ATAGATTAAA AGATGAAGAG ACCAATGAAG 1860ACTCAGGAAG AGATTTGAAA CCACATGAAG ATCAACAAGA TATAAATAAA GATGTGGGTG 1920TGAAGACCTC AGAAAGTACT ACTACAGTAA AAAGCAAAGT TGATGAAAGA GAAAAATATA 1980TGATAGGAAA CTCTGTACAG AAGATCAGTG AACCTGAAGC TGAGATTAGT CCTGGGAGTT 2040TACCAGTGAC TGCAAATATG AAAGCCTCTG AGAACTTGAA GCATATTGTT AACCATGATG 2100ATGTTTTTGA GGAATCTGAA GAACTTTCTT CTGATGAAGA GATGAAAATG GCGGAGATGC 2160GACCACCATT AATTGAAACC TCTATTAACC AGCCAAAAGT CGTAGCACTT AGTAATAACA 2220AAAAAGATGA TACAAAGGAA ACAGATTCTT TATCAGATGA AGTTACACAC AATAGCAATC 2280AGAATAACAG CAATTGTTCT TCTCCATCTC GGATGTCTGA TTCAGTTTCT CTTAATACTG 2340ATAGTAGTCA AGACACCTCA CTCTGCTCTC CAGTGAAACA AACTCATATT GATATTAATT 2400CCAAAATCAG GCAAGAAGAT GAAAATTTTA ACAGCCTTTT ACAAAATGGA GATATTTTAA 2460ACAGTTCAAC AGAGGAAAAG TTCAAAGCTC ATGATAAAAA AGATTTTAAC TTACCTGAAT 2520ATGATTTGAA TGTTGAAGAG CGATTAGTTC TAATTGAGAA AAGTGTTGAC TCAACAGCCA 2580CAGCTGATGA CACTCACAAA TTAGATCATA TCAATATGAA TCTTAATAAA CTTATAACTA 2640ATGATACATT TCAACCAGAG ATCATGGAAA GATCAAAAAC ACAGGATATT GTGCTTGGAA 2700CAAGCTTTTT AAGCATTAAT TCTAAAGAGG AAACTGAGCA CTTGGAAAAT GGAAACAAGT 2760ATCCTAATTT GGAATCCGTA AATAAGGTAA ATGGACATTC TGAGGAAACT TCCCAGTCTC 2820CTAATAGGAC TGAACCACAT GACAGTGATT GTTCTGTTGA CTTAGGTATT TCCAAAAGCA 2880CTGAAGATCT CTCCCCTCAG AAAAGTGGTC CAGTTGGATC TGTTGTGAAA TCTCATAGCA 2940TAACTAATAT GGAGATTGGA GGGCTAAAAA TCTATGATAT TCTTAGTGAT AATGGACCTC 3000AGCAGCCAAG TACAACCGTT AAAATCACAT CTGCTGTTGA TGGAAAAAAT ATAGTCAGGA 3060GCAAGTCTGC CACACTGTTG TATGATCAAC CATTGCAGGT ATTTACTGGT TCTTCCTCAT 3120CTTCTGATTT AATATCAGGA ACAAAGGCAA TTTTCAAGTT TGATTCAAAT CATAATCCCG 3180AAGAGCCAAA TATAATAAGA GGCCCCACAA GTGGCCCACA ATCTGCACCT CAAATATATG 3240GTCCTCCACA GTATAATATC CAATACAGTA GCAGTGCTGC AGTCAAAGAC ACTTTGTGGC 3300ACTCCAAACA AAATCCCCAA ATAGACCATG CCAGTTTTCC TCCTCAGCTC CTTCCTAGAT 3360CAGAGAGCAC AGAAAATCAA AGTTATGCTA AACATTCTGC CAATATGAAT TTCTCTAATC 3420ATAACAATGT TCGAGCTAAT ACTGCATACC ATTTACATCA GAGACTTGGC CCAGCAAGAC 3480ATGGGGAAAT GTGGGCCATC TCACCAAACG ACCGACTTAT TCCTGCAGTA ACTCGAAGTA 3540CAATCCAGCG ACAAAGTAGT GTGTCCTCCA CAGCCTCTGT AAATCTTGGT GATCCAGGCT 3600CTACAAGGCG GGCTCAGATT CCTGAAGGAG ATTATTTATC ATACAGAGAG TTCCACTCAG 3660CGGGAAGAAC TCCTCCAATG ATGCCAGGAT CACAGAGACC CCTTTCTGCA CGAACATACA 3720GCATAGATGG TCCAAATGCA TCAAGACCTC AGAGTGCTCG ACCCTCTATT AATGAAATAC 3780CAGAGAGAAC TATGTCAGTT AGTGATTTCA ATTATTCACG GACTAGTCCT TCAAAAAGAC 3840CAAATGCAAG GGTTGGTTCT GAGCATTCTT TATTAGATCC TCCAGGAAAA AGTAAAGTTC 3900CTCGTGACTG GAGAGAACAA GTACTTCGAC ATATTGAAGC CAAAAAGTTA GAAAAGATGC 3960CTTTGAGTAA TGGACAGATG GGCCAGCCTC TCAGGCCTCA GGCAAATTAT AGTCAAATAC 4020ATCACCCCCC TCAGGCATCT GTGGCAAGGC ATCCCTCTAG AGAACAACTA ATTGATTACT 4080TGATGCTGAA AGTGGCCCAC CAGCCTCCAT ATACACAGCC CCATTGTTCT CCTAGACAAG 4140GCCATGAACT GGCAAAACAA GAGATTCGAG TGAGGGTTGA AAAGGATCCA GAACTTGGAT 4200TTAGCATATC AGGTGGTGTC GGGGGTAGAG GAAACCCATT CAGACCTGAT GATGATGGTA 4260TATTTGTAAC AAGGGTACAA CCTGAAGGAC CAGCATCAAA ATTACTGCAG CCAGGTGATA 4320AAATTATTCA GGCTAATGGC TACAGTTTTA TAAATATTGA ACATGGACAA GCAGTGTCCT 4380TGCTAAAAAC TTTCCAGAAT ACAGTTGAAC TCATCATTGT ACGAGAAGTT TCCTCATAAG 4440CACTGTGGAC AAAAAAAGCG GGGAAGACAG CAAGATTTAT TGGAAGATAC TTACAGGGGA 4500AATTAATATT TTGACTATTT TTATATATAA AGAAGAACTC AAAAAATTAT GTTCAAATTT 4560GTACATTAAT GAAATAATGG ATAAAGGAGA CTGTTGAATT CATACCATAT AAAACTTGTT 4620AGGTTTTTAA ACATAGCAAT CAAGGCTACA AAAACAAACC TGTGTTGTTT TTGTATAGAT 4680TGTAGGTTTA TTTTTGGATT TCATATACAT GACTGAACTG TGTGCAAGGC AATAGTTAGC 4740CTTGATTTTA GCCCAGAGAC AGATGGCAGA GCTATCTCTC TCATAGCTTT TATGCCCTTA 4800TTTTTATTCA ACTGGTATTA ATGTTTTTCT CCTGAAACTA CTTTTTTTGA TGTGGGCAAG 4860AGATTTGAAG TGTTGGCTTT TGCTATGTGC ATATTGAATT GAAGAGTGAG TAGGTGAAGG 4920TGGTGCTGGT GGGTTCACTT TCCAAGGCCA GACTAAAACA GTTATTTTCT ATAAAAATCT 4980GGAAGCAAAG AATGGGGATG GGGAGAGCTA CGTGGTAGTA TGTTTTTATT AGGAGAATAA 5040TGCAATAAAA TATGTAATGT CTTTTTTATA AAGCAAAAAA GACAATAATT GCATTTATGA 5100GCTCGGCAGG ATCTGTTCTT GTCATAGCCA TTGACTATAC ATTTGCTACT GGTGATTCAG 5160TTTTTAATTT TTTAGTCACA GGAAATTTTT AACTCTACTG TAGATGCATG TCCATGCATT 5220TTCTGTGTTA TGGAAATCCA CTGATTTTTT TTTTTTTTTC AAATGGTGGT ACTTGCAATC 5280TGTTTTATAA TTAGTGCTCC ATTTAAATCT AATTTATAAT TTTTATTTTA AGCAGCAAAT 5340GAAACAAAAA TGGCCAGTTT TAAGATTGTG TTGCCTGTAA CACAAAATGT TACGAAGGTT 5400TAGGAAAGCC TCTTTGATTT TTGTTTGGCC TTGCATTGGC CCTTGGTAAA GTAAAAGGAA 5460ACAGTACACT TGGAGCTAGG AAACCAAAGC AAGCTTTGTG AAACTGGCAC AGTGATAGAG 5520AATTGCTGTG GAGAGTTATA GAGCAAAGGG ATGGGTCCTT GAGGCCTGCC AGTGTGTAAA 5580GGTGTTCAAA TAAAGGGCTG TTTCTACAGG TAACATTAAA TGTGAACTCA ACACTTCCAG 5640AGTCTTTAAA GGGTTTCTAT GTGTATCAGT GTAATAGTGT TTTACCACCA ACTGCCTTTC 5700TTTGTTCCTA GTTACTGTAA CAAATATTTG ATGATAGAGG TTTATTAATT TTGTTTATCC 5760AGACCATTAA TTTTATTTGT TTTTGTCTAT GTAATCAAAT AAAATTTGAG TAACATGTAA 5820TGGTAAGGAT TAATGCATGG TTATTTGGAC CAGAAAAAAG TGCCATAGAA GACCAATAAC 5880TGTTTAGTTG AGGCTAGTCT GGAACCTTTC ATTAGAGCAA TATTTGGTTA TTGCACTTCA 5940TTTTTATTTA CTAAGAAATG CAATTTGGGA ATTTTTAATC TGTTATGCTT TGTTTATCAA 6000CCTTGATTTT AATTAAGACT TTTATAAGAC TAGCTTAAAA CACCAACCAA CATTATTTTT 6060GCAAAAGTGA GTTGGACTCA CTTTCCATTC TTGCTAGTCA GAGTAAGTAG GCAGCACTTT 6120TAAAAATATG TGAACTCAAA TATTGCACTT CTTTCAAGAT GTTATCAATT GGTTATTGTA 6180CTGTATAGTT TTAATAATTT TGATTGAAAC CCTTTAACAA CTCTTTGTAA ATTTTAACTC 6240ATTTTAGTTG ATTTTCAGTA CTATTTACAT AGGAATTGAT TTTTATGGAT ATAGTAGAAG 6300AAATGTGCTG TATTTTGATA AAATTCACTT ATTGTATGTG TGTTGTAATC TAAAAAAAAA 6360AAGAATGACA AACAGCTTCT TTAAGACAAA AAAAAAAAAA AAAAAAAAA 6409

What we claim is:
 1. A method of inhibiting proliferation of maligantcells by administration of a Ras-activity inhibitory effective amount ofDNA which encodes the protein Erbin into the maligant cells.
 2. Themethod of claim 1 wherein the amount of Erbin administered is sufficientto provide an intracellular concentration of 1 μM to 100 μM of DNA whichencodes Erbin into the cells.
 3. The method of claim 1 wherein the DNAencoding Erbin is administered via viral vectors.
 4. A method ofevaluating proliferative properties and progression of proliferativeactivities in tumor cells by measurement of Erbin in tumor tissuecomprising: 1) obtaining tumor tissue or cell lysates and 2) measuringthe amount of Erbin in the sample obtained in step
 1. 5. The method ofclaim 4 wherein the amount of Erbin is evaluated by Western blot.
 6. Themethod of claim 4 wherein the amount of Erbin is evaluated byimmunohistochemical analysis.